Establishment of a multicomponent dietary bioactive human equivalent dose to delete damaged Lgr5$^+$ stem cells using a mouse colon tumor initiation model

Abstract

Multi-component therapy has gained interest for its potential to synergize and subsequently lower the effective dose of each constituent required to reduce colon cancer risk. We have previously demonstrated that rapidly cycling Lgr5$^+$ stem cells are exquisitely sensitive to extrinsic dietary factors that modulate colon cancer risk. In the present study, we quantified the dose-dependent synergistic properties of dietary n-3 polyunsaturated fatty acids (PUFA) and curcumin (Cur) to promote targeted apoptotic deletion of damaged colonic Lgr5+ stem cells. For this purpose, both heterogeneous bulk colonocytes and Lgr5$^+$ stem cells were isolated from Lgr5-EGFP-IRES-CreER$^{\rm{T2}}$ knock-in mice injected with azoxymethane (AOM). Isolated cells were analyzed for DNA damage ($\gamma$H2AX), apoptosis (cleaved caspase-3) and targeted apoptosis (both γH2AX and cleaved caspase-3) at 12 hr post AOM injection. Comparison of the percentage of targeted apoptosis in Lgr5$^+$ stem cells (GFP$^{\rm{high}}$) across a broad bioactive dose range revealed an ED50 of 16.0 mg/d n-3 PUFA + 15.9 mg/d Cur. This corresponded to a human equivalent dose (HED) of 3.0 g n-3 PUFA + 3.0 g Cur. In summary, our results provide evidence that a low dose (n-3 PUFA + Cur) combination diet reduces AOM-induced DNA damage in Lgr5$^+$ stem cells and enhances targeted apoptosis of DNA damaged cells, implying that a lower HED can be utilized in future human clinical trials.